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Novel constructed high-yield fumaric acid gene engineering bacterium and method for producing fumaric acid thereby

A technology of genetically engineered bacteria and fumaric acid, applied in the field of bioengineering, can solve problems such as low production intensity, high price, and slow growth of mold

Inactive Publication Date: 2008-08-13
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic conversion method that appeared in the mid-1990s generally uses maleic acid as a substrate to prepare fumaric acid under the action of maleic acid isomerase, but the supply of raw material maleic acid is insufficient and expensive
However, the mold growth rate is slow, and the product production intensity is low

Method used

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  • Novel constructed high-yield fumaric acid gene engineering bacterium and method for producing fumaric acid thereby
  • Novel constructed high-yield fumaric acid gene engineering bacterium and method for producing fumaric acid thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] This example illustrates the process of using homologous recombination technology to knock out the fumarate reductase frd gene in parent Escherichia coli NZN111 to obtain apramycin-resistant strains.

[0035] 1. Using LB medium, cultivate E. coli NZN111 to OD at 37°C under aerobic conditions 600 = 0.4-0.6, prepared into electro-competent state.

[0036]2. Electrotransform the recombinant plasmid into competent E. coli NZN111. The electric shock conditions are: 200Ω, 25μF, electric shock voltage 2.3kv, electric shock time 4~5ms. After the electric shock, the cells were quickly added to the pre-cooled 1mL SOC medium, cultured at 150r / min and 30°C for 1h, and then spread on the LB medium plate with ampicillin (amp) to select the positive transformant NZN111 (pKD46).

[0037] 3. Add 10mM L-arabinose to LB medium, induce plasmid pKD46 to express lambda recombinase at 30°C, and make it electrocompetent.

[0038] 4. Using the apramycin resistance gene with FRT sites on both sides ...

Embodiment 2

[0050] This example illustrates the process of constructing an expression plasmid that overexpresses malic enzyme, restoring the ability of the recombinant strain to metabolize glucose under anaerobic conditions, and obtaining the strain Escherichia coli JM125.

[0051] 1. The process of constructing an expression plasmid that overexpresses malic enzyme includes:

[0052] (1) Synthesis of primers with Nco I and HindIII restriction sites, upstream primers:

[0053] 5’-CATGCCATGGATATTCAAAAAAGAGTGAGTG-3’, downstream primer:

[0054] 5’-CCCAAGCTTTTAGATGGAGGTACGGC-3’

[0055] (2) Using E. coli K12 as template, colony PCR, the reaction conditions were 95°C, 1.5min, 63°C, 1.0min, 72°C, 1.5min, 35 cycles in total. After purifying the amplified sfcA gene, the expression plasmid pTrc99a was digested with Nco I and Hind III and ligated to obtain the recombinant plasmid pTrc99a-sfcA.

[0056] 2. The plasmid pTrc99a-sfcA was introduced into Example 1 to eliminate the competence of the apramyci...

Embodiment 3

[0058] This example illustrates the comparison between the newly constructed recombinant Escherichia coli JM125 and the starting strain NZN111 for fermentation of acid production capacity.

[0059] Escherichia coli NZN111 (CGSC7726), when introduced into the plasmid pTrc99a-sfcA, overexpressed the malic enzyme gene, restored the ability of NZN111 to metabolize glucose under anaerobic conditions, and had a high yield of succinic acid accumulation. The constructed recombinant Escherichia coli JM125, when grown under anaerobic conditions, produces a mixed acid of fumaric acid, succinic acid and acetic acid, of which fumaric acid is the main product. In order to investigate the effect of the reduction of fumarate reductase activity on acid production, a two-stage fermentation mode was adopted, and 1% (v / v) inoculum amount was inserted from the cryotube into the Erlenmeyer flask. 600 To about 0.8~1.0, induce to OD with 0.7mM IPTG 600 = About 3, transfer 10% of the inoculum to the serum...

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Abstract

The invention discloses a method for constructing genetic engineering bacterial strain Escherichia coli JM125 producing boletic acid and method for producing acid. The constructing process comprises inactivation or out-knocking enzymes in facultative microbiology which has abilities of reduction boletic acid and degrading acetonic acid, and exceeding expressing NAD regeneration. The following steps of two-stage fermentation experiment using constructed Escherichia coli are (1) activating bacillus coli is transferred to enrichment medium and cultured to improve biomass. (2) The treated bacterium liquid membrane obtained by aerobic cultivation is transferred into LB culture medium which contains 20 to 100g / L amylaceum and certain concentration carbonates to produce fumaric acid by anaerobic fermentation.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, relates to the construction of a fumaric acid-producing recombinant strain, and also relates to a method for fermentative production of fumaric acid by using the strain. Background technique [0002] Fumaric acid (also known as fumaric acid), as an important four-carbon platform compound, has a wide range of uses in the pharmaceutical, food, and surfactant industries. It can be used to produce L-day through enzyme-catalyzed conversion, esterification, and hydrogenation. Four-carbon compounds such as aspartic acid, malic acid, succinic acid, maleic acid, 1,4-butanediol, γ-butyrolactone and tetrahydrofuran. At present, fumaric acid is mainly produced through the isomerization of maleic anhydride and furfural oxidation. Due to the high cost and environmental pollution of chemical synthesis of fumaric acid, the wide application of fumaric acid as a basic chemical raw material is restricted. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/09C12P7/46C12R1/19
Inventor 姜岷马江锋王益娜于丽黄秀梅
Owner NANJING UNIV OF TECH
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